Approximately 8,000 children in the USA are born each year with some form of hearing loss severe enough to require special services. Half of these are genetic in origin covering several hundred different regions in the genome where mutations affect the hearing process. One example of genetic predisposition makes some people more likely to develop hearing loss after taking certain antibiotic medications. Infants may also be born with hearing loss caused by a viral infection like cytomegalovirus (CMV) that was acquired during pregnancy. Newborns in the neonatal intensive care units are more likely to suffer from hearing loss as a toxic side effect of the antibiotics which are frequently administered. It is very challenging, while extremely important, to understand the cause of the disease and its treatment. The current approach of audiometric screening doesn't provide the genetic etiology, and the single gene sequencing method used to compliment this is costly, time consuming and impractical, requiring a large blood sample that cannot be easily obtained safely from an infant. We are developing a test using massively parallel DNA sequencing [Next Generation Sequencing (NGS)] methodology that can address the deficiencies of current testing and be used as a routine second-tier newborn screening tool in high-risk patients identified by current audiometric hearing screening. The test can also be used as a diagnostic for individuals with post lingual hearing loss for the identification of disease-causing loci. Such NGS sequencing panel-based screening and diagnostic methodologies are currently not available from a commercial company or for addressing the needs of newborns. Such commercial NGS-based diagnostic methods can be rapidly expanded throughout the country as a service, permitting quick diagnostic decisions, and have the potential to reduce healthcare costs by avoiding diagnostic delays. The newborn hearing screening protocols that are currently in use can result in over half of newborns who fail the screening test to be lost to follow-up and do not address the problem of congenital infections that cause hearing loss. Our first Specific Aim is to make this test accurate and appropriate for newborns and therefore we will focus on the right specimen collection strategy so the highest sensitivity and specificity is reached. In our second Specific Aim we will focus on a comprehensive gene panel addressing all relevant genetic loci and CMV specific panel in a single test and make it robust and accurate. The resulting prototype will be a valuable second tier-screening and diagnostic tool that will lead to a test that will address the needs of these high-risk infants. It will help healthcare providers, patients, and thei families to understand the precise cause of the hearing loss, and to plan accordingly. Our test is unique in that it simultaneously screens and/or diagnoses hundreds of these conditions at once from a single sample, providing more comprehensive information to families and their physicians and yet affordable and provide access to the associated data.